Modified nucleotides of tRNAPro restrict interactions in the binary primer/template complex of M-MuLV.
In all retroviruses, reverse transcription is primed by a cellular tRNA, which is base-paired through its 3'-terminal 18 nucleotides to a complementary sequence on the viral RNA genome termed the primer binding site (PBS). Evidence for specific primer-template interactions in addition to this standard interaction has recently been demonstrated for several retroviruses. Here, we used chemical and enzymatic probing to investigate the interactions between Moloney murine leukemia virus (M-MuLV) RNA and its natural primer tRNAPro. The existence of extended interactions was further tested by comparing the viral RNA/tRNAPro complex with simplified complexes in which viral RNA or tRNA were reduced to the 18 nt of the PBS or to the complementary tRNA sequence. These data, combined with computer modeling provide important clues on the secondary structure and three-dimensional folding of the M-MuLV RNA/tRNAPro complex. In contrast with other retroviruses, we found that the interaction between tRNAPro and the M-MuLV RNA template is restricted to the standard PBS interaction. In this binary complex, the viral RNA is highly constrained and the rest of tRNAPro is rearranged, with the exception of the anticodon arm, leading to a very compact structure. Unexpectedly, when a synthetic tRNAPro lacking the post-transcriptional modifications is substituted for the natural tRNAPro primer, the interactions between the primer and the viral RNA are extended. Hence, our data suggest that the post-transcriptional modifications of natural tRNAPro prevent additional contacts between tRNAPro and the U5 region of M-MuLV RNA.
Fossé
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P
Institut de Biologie Moléculaire et Cellulaire, 15 rue Descartes, Strasbourg cedex, 67084, France.
Mougel
M
M
Keith
G
G
Westhof
E
E
Ehresmann
B
B
Ehresmann
C
C
D008958:Models, Molecular; D008979:Moloney murine leukemia virus / Q000737:chemistry* / Q000378:metabolism; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D009711:Nucleotides / Q000737:chemistry; D012313:RNA / Q000737:chemistry / Q000378:metabolism; D012361:RNA, Transfer, Pro / Q000737:chemistry* / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry* / Q000378:metabolism; D013698:Templates, Genetic
D009711:Nucleotides; C034477:RNA primers; D012361:RNA, Transfer, Pro; D012367:RNA, Viral; D012313:RNA